This invention relates to microbial production of chemicals. More particularly, the invention relates to methods for microbial production of epoxides and hydroxylated chemical feedstocks in a multiphase reactor using gaseous inputs as precursors and for regeneration of reducing equivalents.
Methane monooxygenase (EC 1.14.13.25) is a multicomponent enzyme, produced by methanotrophic bacteria, which catalyzes the incorporation of atmospheric oxygen into methane to form methanol. This is the first in a series of reactions that ultimately provide energy and carbon to the cell. Methanol dehydrogenase further oxidizes methanol to form formaldehyde. Although reducing equivalents are consumed initially by the monooxygenase, the methanotrophic bacteria regenerate NADH by subsequent oxidations mediated by formaldehyde and formate dehydrogenases, respectively, the terminal product being CO2 (for an excellent review, see J. D. Lipscomb, Biochemistry of the Soluble Methane Monooxygenase, 48 Annu. Rev. Microbiol. 371-399 (1994)).
Studies of methane monooxygenase refer to both a soluble (sMMO) and membrane-bound or particulate (pMMO) enzyme. The prevalent form in type II methanotrophs, such as Methylosinus trichosporium OB3b, and type X methanotrophs, such as Methyloccus capsulatus, is regulated in some fashion by the concentration of copper in the growth medium. K. J. Burrows et al., Substrate Specificities of the Soluble and Particulate Methane Mono-oxygenases of Methylosinus trichosporium OB3b, 130 J. Gen Microbiol. 3327-3333 (1984); S. H. Stanley et al., Copper Stress Underlies the Fundamental Change in Intracellular Location of Methane Monooxygenase in Methane-oxidizing Microorganisms: Studies in Batch and Continuous Culture, 5 Biotechnol. Lett. 487-492 (1983). Type I methanotrophs such as Methylomonas methanica typically express only pMMO, although one recent isolate has been found to be an exception to that generalization. S. -C. Koh et al., Soluble Methane Monooxygenase Production and Trichloroethylene Degradation by a Type I Methanotroph, Methylomonas methanica 68-1, 59 Appl. Environ. Microbiol. 960-967 (1993).
Components of sMMO from Methylosinus trichosporium OB3b that retained a high specific activity were initially purified by B. G. Fox et al., Methane Monooxygenase from Methylosinus trichosporium OB3b. Purification and Properties of a Three-component System with High Specific Activity from a Type II Methanotroph, 264 J. Biol. Chem. 10023-10033 (1989). The enzyme comprises a 245 kDa hydroxylase containing a hydroxo-bridged dinuclear iron cluster at the active site of methane oxidation, a 15.8-kDa component B, and a 38.4-kDa iron-sulfur reductase with a flavin prosthetic group.
Although not supporting growth, sMMO and pMMO will, in addition to methane, adventitiously oxidize a variety of alkanes and alkenes. C. T. Hou et al., Microbial Oxidation of Gaseous Hydrocarbons: Epoxidation of C2 to C4 n-alkenes by Methylotrophic Bacteria, 38 Appl. Environ. Microbiol. 127-134 (1979); D. I. Stirling et al., A Comparison of the Substrate and Electron-donor Specificities of the Methane Monooxygenases from Three Strains of Methane-oxidizing Bacteria, 177 J. Biochem. 361-364 (1979); H. Dalton, Oxidation of Hydrocarbons by Methane Monooxygenases from a Variety of Microbes, 26 Adv. Appl. Microbiol. 71-87 (1980). The substrate range of sMMO, however, also includes aromatic and alicyclic compounds, K. J. Burrows et al., supra; J. Colby et al., The Soluble Methane Monooxygenase of Methylococcus capsulatus (Bath). Its Ability to Oxygenate n-Alkanes, n-Alkenes, Ethers, and Alicyclic, Aromatic, and Heterocyclic Compounds, 165 J. Biochem. 395-402 (1977), ethers and heterocyclic compounds, J. Colby et al., supra, and halogenated aromatics and alkenes, J. Green and H. Dalton, Substrate Specificity of Soluble Methane Monooxygenase. Mechanistic Implications. 264 J. Biol. Chem. 17698-17703 (1989).
Currently, there is considerable interest in enzyme function in non-aqueous solvents (for reviews, see J. S. Dordick, Enzymatic Catalysis in Monophasic Organic Solvents, 11 Enzyme Microb. Technol. 194-211 (1989); P. Nickolova and O. P. Ward, Whole Cell Biocatalysis in Nonconventional Media, 12 J. Ind. Microbiol. 76-86 (1993); G. J. Salter and D. B. Kell, Solvent Selection for Whole Cell Biotransformations in Organic Media, 15 Crit. Rev. Biotechnol. 139-177 (1995)). Several beneficial and unexpected modifications of typical enzyme behavior were noted in such systems. These include enhanced enzyme activity, R. Batra and M. N. Gupta, Enhancement of Enzyme Activity in Aqueous-organic Solvent Mixtures, 16 Biotechnol. Lett. 1059-1064 (1994), increased thermostability, and alterations in substrate specificity, A. Zaks and A. M. Klibanov, Enzymatic Catalysis in Organic Media at 100xc2x0 C., 224 Science 1249-1251 (1984).
Recent efforts have concentrated on applications that may be less amenable to strictly aqueous approaches, since the substrates are largely insoluble in water. Examples of such an approach include degradation of sparingly soluble xenobiotics, M. Ascon-Cabrera and J. -M. Lebeault, Selection of Xenobiotic-degrading Microorganisms in a Biphasic Aqueous-organic System, 59 Appl. Environ. Microbiol. 1717-1724 (1993), petroleum fuel desulfurization, W. R. Finnerty, Organic Sulfur Biodesulfurization in Non-aqueous Media, 72 Fuel 1631-1634 (1993), and coal modification or solubilization, E. S. Olson et al., Non-aqueous Enzymatic Solubilization of Coal-derived Materials, 72 Fuel 1687-1693 (1993); C. D. Scott et al., The Chemical Modification of Enzymes to Enhance Solubilization in Organic Solvents for Interaction with Coal, 72 Fuel 1695-1700 (1993).
Production of propylene oxide from propylene for use as a chemical feedstock has been investigated using immobilized whole cells. L. E. S. Brink and J. Tramper, Production of Propene Oxide in an Organic Liquid-phase Immobilized Cell Reactor, 9 Enzyme Microb. Technol. 612-618 (1987); C. T. Hou, Propylene Oxide Production from Propylene by Immobilized Whole Cells of Methylosinus sp. CRL-31 in a Gas-solid Bioreactor, 19 Appl. Microbiol. Biotechnol. 1-4 (1984).
T. R. Clark and F. F. Roberto, Methylosinus trichosporium OB3b Whole-cell Methane Monooxygenase Activity in a Biphasic Matrix, 45 Appl. Microbiol. Biotechnol. 658-663 (1996), demonstrated soluble methane monooxygenase activity in a two-phase (biphasic) matrix comprising a buffered aqueous phase and 2,2,4-trimethylpentane (isooctane) using reconstituted whole-cell preparations of lyophilized Methylosinus trichosporium OB3b. The rate of conversion of gaseous propylene to propylene oxide, a non-metabolized liquid, was used as the primary measure of enzymatic activity. Appreciable soluble methane monooxygenase activity was detected when the volume of the aqueous phase represented at least 1% of the total volume, although the initial rate of product formation did increase as the volume of the aqueous phase increased. In comparison to the aqueous system, the specific rate and yields in the biphasic system were much less sensitive to increases in the concentrations of formate and protein (i.e., the methane monooxygenase). There was some evidence, however, that the enzyme system was more stable in the biphasic matrix, since the rate of propylene oxide formation remained linear for an extended period of time. V(app.) in the biphasic system decreased by a factor of 0.6 relative to the same parameter in the aqueous system. Conversely, Km(app.) for propylene was 1.6 times greater in the biphasic system. Hence, the apparent catalytic efficiency in the aqueous system was four times that in the biphasic system, as indicated by a decrease in the corresponding ratios of V(app.) to Km(app.).
In view of the foregoing, it will be appreciated that providing a method for microbial production of epoxides and hydroxylated hydrocarbons would be a significant advancement in the art.
It is an object of the present invention to provide a method for microbial production of epoxides and hydroxylated hydrocarbons in a continuous production format.
It is also an object of the invention to provide a method for microbial production of epoxides and hydroxylated hydrocarbons that utilizes gaseous inputs and produces liquid products.
It is another object of the invention to provide a method for microbial production of epoxides and hydroxylated hydrocarbons that results in regeneration of reducing equivalents.
It is still another object of the invention to provide a method for microbial production of epoxides and hydroxylated hydrocarbons that uses a liquid phase comprising (i) a non-polar, non-aqueous solvent or mixture of miscible non-polar, non-aqueous solvents and (ii) an aqueous phase.
It is yet another object of the invention to provide a method for microbial production of epoxides and hydroxylated hydrocarbons wherein a hydrated biocatalyst can be immobilized on a non-porous support material and submerged in the non-polar, non-aqueous solvent phase.
It is a further object of the invention to provide a method for microbial production of epoxides and hydroxylated hydrocarbons wherein non-aqueous solvents have low vapor pressures, much higher boiling points than reaction products, and are non-inhibitory to methane oxidizing microorganisms.
It is a still further object of the invention to provide a method for microbial production of epoxides and hydroxylated hydrocarbons wherein water and other polar molecules, such as reaction products, are insoluble in the non-aqueous solvents.
It is another object of the invention to provide a method for microbial production of epoxides and hydroxylated hydrocarbons wherein the gaseous substrates are readily soluble in the non-aqueous solvents.
It is still another object of the invention to provide a method for microbial production of epoxides and hydroxylated hydrocarbons wherein the products are polar and readily soluble in the aqueous phase.
It is yet another object of the invention to provide a method for microbial production of epoxides and hydroxylated hydrocarbons wherein the density of the non-aqueous phase is much greater than the density of the aqueous phase such that a stable, self-maintaining phase separation is obtained.
These and other objects can be addressed by providing a method for microbial production of an oxygenated derivative of a methane-monooxygenase substrate comprising:
(a) incubating a reconstituted lyophilized whole cell enzyme preparation of methanotrophic bacteria containing methane monooxygenase and hydrogenase in a bioreactor, said bioreactor comprising a biphasic liquid phase comprising a major amount of a non-polar non-aqueous solvent and a minor amount of a polar aqueous medium, a non-aqueous solvent circulation circuit for circulating said non-polar non-aqueous solvent through the bioreactor, and an aqueous medium circulation circuit for circulating the polar aqueous medium through the bioreactor in a counter-current manner as compared to the non-polar non-aqueous solvent;
(b) continuously dissolving effective amounts of oxygen gas, hydrogen gas, and a gaseous substrate in the non-polar non-aqueous solvent, wherein the gaseous substrate is readily soluble in the non-polar non-aqueous solvent and is susceptible to oxidation by the methane monooxygenase to result in the oxygenated derivative thereof, wherein the oxygenated derivative is a polar liquid;
(c) circulating the non-polar non-aqueous solvent in close proximity to the reconstituted lyophilized whole cell enzyme preparation of methanotrophic bacteria containing methane monooxygenase and hydrogenase such that the methane monooxygenase oxidizes the gaseous substrate into the oxygenated substrate;
(d) circulating the polar aqueous medium in a counter-current manner through the circulating non-polar non-aqueous solvent such that the oxygenated substrate is partitioned into the polar aqueous medium; and
(e) removing and recovering the oxygenated substrate from the polar aqueous medium and recycling the non-polar non-aqueous solvent and the polar aqueous medium.
Many substrates and products can be used in this method, but a preferred substrate is propylene, and the resulting preferred oxygenated derivative is propylene oxide. Preferred methanotrophic bacteria according to the invention are selected from the group consisting of Methylosinus, Methyloccus, and Methylomonas, and mixtures thereof. Especially preferred methanotrophic bacteria are Methylosinus trichosporium OB3b. Preferred non-polar non-aqueous solvents according to the invention are isooctane, hexane, silicone oil, hexadecane, fluorocarbons, and mixtures thereof.
Another preferred embodiment of the invention comprises a method for regenerating reducing equivalents in a bioreactor configured for utilizing reducing equivalents in a methane-monooxygenase-catalyzed reaction for oxidizing a substrate into an oxygenated product comprising:
(a) incubating a reconstituted lyophilized whole cell enzyme preparation of methanotrophic bacteria containing methane monooxygenase and hydrogenase in the bioreactor, the bioreactor comprising a liquid phase comprising a major amount of a non-polar non-aqueous solvent and a minor amount of a polar aqueous medium; and
(b) continuously dissolving an effective amount of hydrogen gas in the non-polar non-aqueous solvent, such that the hydrogen gas and NAD+ are converted by the hydrogenase into NADH, thereby regenerating reducing equivalents.
Still another preferred embodiment of the invention comprises a method for preparing a lyophilized preparation of methanotrophic bacteria for use as a biocatalyst comprising:
(a) culturing the methanotrophic bacteria, concentrating the resulting cells, resuspending the concentrated cells, and then chilling the resuspended cells at about 0xc2x0 C.;
(b) freezing the chilled cells at about xe2x88x9250xc2x0 C. using a shell freezer;
(c) further cooling the frozen cells in a liquid nitrogen bath and then freeze drying the resulting cells using a lyophilizer for a period sufficient to obtain a powdered cell preparation; and
(d) storing the powdered cell preparation under refrigeration.
A still further preferred embodiment of the invention comprises a method for propagating methanotrophic bacteria comprising culturing the bacteria in a liquid medium comprising a major amount of a non-aqueous polar solvent and a minor amount of a known methanotrophic bacterial growth medium.